Isolation of antigen-specific antibodies has been achieved through a variety of methods, including screening of phage-display recombinant antibody (scFv) libraries. Yeast-display recently emerged as an efficient alternative strategy for scFv identification that offers several advantages over prokaryotic systems, including superior sampling of the immune antibody repertoire; post-translational modifications (glycosylation) due to the eukaryotic expression; faster and more controlled flow cytometry-based selection compared to solid phase panning; and absence of growth bias, as recombinant proteins are displayed at the yeast cell surface only during the induction step in the presence of galactose. Yet, previously reported yeast libraries have been severely limited in size, with typically less than 1×105 transformants per microgram of DNA for commonly used strains, resulting in insufficient diversity and potential for yielding high affinity antibodies. Additionally, with existing methods, transfer of scFv from displayed to secreted forms has often resulted in loss of antigen specificity and/or affinity, requiring additional time-consuming and costly steps, including in vitro maturation of scFv sequence and/or recloning of scFv fused to immunoglobulin (Ig) constant regions. The mechanisms underlying loss of scFv function include changes in scFv conformation and post-translational modification due to different expression systems for displayed and secreted forms.
It was hypothesized that the use of electroporation combined with buffer modifications could remove obstacles contributed by poor yeast transformation efficiency. In addition, we hypothesized that only one expression system (Saccharomyces cerevisiae) for both scFv display and secretion could eliminate changes in scFv post-translational modifications, while keeping the advantages of an eukaryotic system for the expression of high-affinity antibodies. It was also hypothesized that if both displayed and secreted scFv were modified only at the N-terminus, which binds to the yeast surface or to secondary reagents, respectively, conformational changes would be minimized during the shift from displayed to secreted forms. To test this hypotheses, a previously generated M13 bacteriophage display human scFv library was transferred through homologous recombination into our novel vector pAGA2 for yeast-display. This yielded a 1×109-member yeast scFv display library, which was then screened in two steps using two novel complementary yeast systems. The first was engineered to permit scFv surface expression as a fusion with an Aga2 protein to N-termini for convenient high-throughput screening by flow sorting. The second was engineered to permit rapid transformation into yeast-secreted soluble scFvs fused to N-termini to an IgA hinge and an enzymatically biotinylatable site for in vitro and in vivo validation. Such scFv are called “biobodies” after targeted biotinylation by yeast mating. As proof of principle, we used this novel platform to screen for scFv against the tumor marker endosialin/tumor endothelial marker 1 (TEM1) or CD248, an attractive target for antibody-based tumor diagnosis and therapy. Several high-affinity TEM1-specific yeast-secreted scFvs and biobodies were isolated and characterized for binding to human and murine TEM1 in vitro and in vivo. The highest affinity anti-TEM1 biobody-78 (Kd 4 nM) was able to bind both murine and human TEM1 and selectively targeted TEM1-expressing tumor cells in a novel in vivo mouse model of orthotopic ovarian cancer. This streamlined approach for rapid identification of high affinity reagents suitable for in vivo use paves the way for the high throughput development of novel antigen-targeted theranostics.